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  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

• the field of this invention is Listeria species, e.g., Listeria monocytogenes, particularly recombinant strains of Listeria species, and methods for their construction and use.
• Listeria species e.g., Listeria monocytogenes, particularly recombinant strains of Listeria species, and methods for their construction and use.
• Background of the Invention The use of vaccines is a cost-effective medical tool for the management of infectious diseases, including infectious diseases caused by bacteria, viruses, parasites, and fungi. In addition to effecting protection against infectious diseases, effort is also being expended to develop vaccines that stimulate the host's immune system to intervene in tumor growth. Host immune responses include both the humoral immune response involving antibody production and the cell-mediated immune response. Protective immunization via vaccine has usually been designed to induce the formation of humoral antibodies directed against infectious agents, tumor cells, or the action of toxins.
• live infectious agents include live infectious agents which may gain access to the cytoplasm of host cells where the proteins encoded by these agents are processed into epitopes which when presented to the cellular immune system, induce a protective response.
• Microorganisms particularly Salmonella and Shigella, which have been attenuated using a variety of mechanisms have been examined for their ability to encode and express heterologous antigens.
• Such bacteria may be useful as live attenuated bacterial vaccines which serve to induce a cellular immune response directed against a desired heterologous antigen.
• this bacterial-based vector has significant advantages over other recombinant vaccine delivery systems.
• safety concerns regarding the use in vivo of this bacterial vaccine vector remain an important issue.
• the use of the most common wild-type strain of Listeria, L. monocytogenes can be accompanied by severe side effects, including the development of listeriosis in the inoculated animal.
• This disease which is normally food-borne, is characterized by meningitis, septicemia, abortion and often a high rate of mortality in infected individuals. While natural infections by L. monocytogenes are fairly rare and may be readily controlled by a number of antibiotics, the organism may nevertheless be a serious threat in immunocompromised or pregnant patients.
• the subject bacteria are characterized by having a mutation in a gene chosen from the IplA gene and the hly gene.
• the subject bacteria find use in a variety of applications, where representative applications of interest include, but are not limited to: (a) use of the subject bacteria as adjuvants; (b) use of the subject bacteria as delivery vectors for introducing macromolecules into a cell; (c) use of the subject bacteria as vaccines for eliciting or boosting a cellular immune response; etc.
• FIGS. 2A-2F Growth of the Cytotoxic Mutants in J774 macrophage-like cells and C57BL/6 Mice.
• 2A Colony forming units found within a monolayer of J774 cells on a 12 mm glass coverslip, at the indicated time, in the presence of the extracellular antibiotic gentamicin added 1 hour post-infection. Data represents the mean values derived from 3 coverslips.
• 2B Colony forming units found within a monolayer of J774 cells on a 12 mm glass coverslip, at the indicated time, with gentamicin treatment from 1 hour to 2 hours post-infection. Data represents the mean values derived from 3 coverslips.
• Figures 4A -4C The Greater the Cytotoxicity, the Greater the Virulence Defect.
• a competitive index was established by injecting both wild-type bacteria and eiythromycin-resistance-marked mutants into C57BL/6 mouse tail-veins.
• Competitive Index ratios were established in the spleen and liver by competing 4A) wild-type versus LLO L461T Erm, 4B) wild-type versus LLO S44A Erm, and 4C) wild-type versus LLO S44A L461T Erm.
• the y-axis indicates the ratio of the number of mutant CFU divided by the CFU of wild-type bacteria isolated from the spleen or liver of mice at the indicated time points on a log scale.
• FIGS 5A & 5B Cytotoxic mutants are more sensitive to Gentamicin.
• 5A 1 x 10 5 CFU wild-type bacteria were injected into the tail vein of C57BL/6 mice. 1 mg of gentamicin was injected subcutaneously, and at the indicated time the liver and spleen were removed, homogenized, and plated to determine CFU in each organ. Error bars indicate standard deviation from a minimum of 7 mice.
• 5B Competitive indexes were established at 48 hours as described in Figure 4. Data points labeled as "+ RB6 gent" were injected with RB6-8C5 monoclonal antibodies 6 hours pre-infection as well as 1 mg gentamicin sulfate subcutaneously 6 hours before organ harvesting. SALT indicates data from the LLO S44A L461T strain.
• FIGS. 6A & 6B The Virulence Defect is not Due to Defects in Cell-to-Cell Spread.
• Competitive indexes were performed as described in Figure 4.
• ⁇ ActA all strains including the reference strain, secreting wild- type LLO, contained an in-frame deletion in actA that eliminated actin-based motility.
• 6A "24 hr" and "48 hr” indicate the time of organ harvest.
• the data points represent the ratio of erythromycin-resistant LLO L461T bacteria divided by the CFU of wild-type bacteria in the spleen and liver.
• the subject bacteria are characterized by having a mutation in a gene chosen from the IplA gene and the hly gene.
• the subject bacteria find use in a variety of applications, where representative applications of interest include, but are not limited to: (a) use of the subject bacteria as adjuvants; (b) use of the subject bacteria as delivery vectors for introducing macromolecules into a cell; (c) use of the subject bacteria as vaccines for eliciting or boosting an antigen-specific humoral or cellular immune response; etc.
• the subject attenuated bacteria are reviewed first in greater detail, followed by a review of representative applications in which the subject vectors and methods find use.
• the subject invention provides attenuated Listeria bacteria.
• attenuated describes the diminution in the ability of the subject bacteria to cause disease in an animal as a whole, e.g., as measured by the LD 5 o of the bacteria, as described below. More specifically, the pathogenic characteristics of the attenuated Listeria strain, as viewed from the vantage of the host animal as a whole (as opposed to a cell based perspective) have been lessened compared with wild-type Listeria, although the attenuated Listeria is capable of growth and maintenance in culture.
• bacteria are considered to be attenuated if, upon the intravenous inoculation of Balb/c mice (as described in the experimental section, below), the lethal dose at which 50% of inoculated animals survive (LD 5 o) is increased above the LD 5 0 of wild-type Listeria by at least about 10-fold, such as by at least about 100-fold, including by at least about 1 ,000 fold, where in certain embodiments the magnitude of increase is at least about 10,000 fold, such as at least about 100, 000-fold, as determined using the assay employed in the experimental section below.
• An attenuated strain of Listeria according to the subject invention is thus one which does not kill an animal to which it is administered, or is one which kills the animal only when the number of bacteria administered is vastly greater than the number of wild type non-attenuated bacteria which would be required to kill the same animal.
• Attenuated species according to the subject invention are ones that exhibit a decreased virulence compared to their corresponding wild type strain in the Competitive Index Assay as described in Auerbach et al., "Development of a Competitive Index Assay To Evaluate the Virulence of Listeria monocytogenes actA Mutants during Primary and Secondary Infection of Mice," Infection and Immunity, September 2001 , p. 5953-5957, Vol. 69, No. 9.
• mice are inoculated with test and reference, e.g., wild- type, strains of bacteria.
• a given bacterial strain is considered to be less virulent if its abundance in the spleen is at least about 50-fold, or more, such as 70-fold or more less than that observed with the corresponding wild-type strain, and/or its abundance in the liver is at least about 10-fold less, or more, such as 20-fold or more less than that observed with the corresponding wild-type strain.
• bacteria are considered to be less virulent if they show abortive replication in less than about 8 hours, such as less than about 6 hours, including less than about 4 hours, as determined using the assay described in Jones and Portnoy, Intracellular growth of bacteria. (1994b) Methods Enzymol. 236:463-467.
• bacteria are considered to be attenuated or less virulent if, compared to wild-type, they form smaller plaques in the plaque assay employed in the Experimental Section, below, where cells, such as murine L2 cells, are grown to confluency, e.g., in six-well tissue culture dishes, and then infected with bacteria.
• DME-agar containing gentamicin is added and plaques are grown for a period of time, e.g., 3 days. Living cells are then visualized by adding an additional DME-agar overlay, e.g., containing neutral red (GIBCO BRL) and incubated overnight.
• an additional DME-agar overlay e.g., containing neutral red (GIBCO BRL) and incubated overnight.
• the magnitude in reduction in plaque size observed with the attenuated mutant as compared to the wild-type is, in certain embodiments, 10%, including 15%, such as 25% or more.
• the subject bacteria may be any Listeria species that is rendered attenuated according to the subject invention.
• strains of Listeria other than L. monocytogenes may be used for the generation of attenuated mutants according to the present invention.
• the Listeria strain is L. monocytogenes.
• the subject bacteria are cytotoxic.
• a particular strain of bacteria is considered to be cytotoxic if it compromises its host cell in a period of less than about 8 hours, sometimes less than about 6 hours, e.g., in less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about two hours, or less than about 1 hour, as determined using the cytotoxicity assay described below.
• Representative cytotoxic bacterial strains according to the subject invention include those hly mutant stains described below.
• the subject bacteria comprise a mutated hly gene, by which is meant that the bacteria comprise an hly gene where the coding sequence of the gene has been altered to encode an LLO product whose amino acid sequence differs from wild type LLO by at least one residue, e.g, by missing the at least one residue, by having a substitute for at least one residue, etc.
• the encoded product is a deletion mutant, by which is meant that one or more residues found in the wild type protein are absent or missing in the mutant polypeptide, where the missing residues are not replaced by substitute residues.
• the encoded product is a point mutant, by which is meant that one or more residues of the wild type protein, which may or may not be adjacent to one another, are substituted with a different residue.
• the mutant hly gene is one that encodes a mutant LLO product that has more hemolytic activity at neutral pH than the wild type LLO protein, where the hemolyticity is determined using the assay described in Glomski et al., J. Cell Biol. (March 18, 2002) 156:1029-1038 and the Experimental Section, below. As measured by this assay, the encoded mutant LLO protein of the bacteria of these embodiments is at least about 2-fold, sometimes at least about 5-fold and sometimes at least about 10-fold more hemolytic than the wild type LLO protein.
• the mutation of the hly gene is one that encodes a point mutant product, such that one or more residues in the encoded product differs from the corresponding residue in the wild type protein.
• a residue falling between 450 and 470, often between 455 and 465 is substituted, where in certain embodiments the substituted residue is residue 461.
• the L at position 461 is substituted with a non-L residue, where the substituting residue may be T, N, Q, S etc, but is T in certain embodiments.
• the mutation is found in domain 3, or a residue that interacts with a residue in domain 3.
• the mutant hly gene includes a mutation in the PEST-like sequence encoding domain of the gene.
• the PEST-like sequence of the encoded product is found in the N-terminal 75 residues of the LLO protein, and more specifically in the N-terminal 60 residues of the LLO protein, and more precisely between residues 34 and 59.
• the mutation of the PEST-like sequence encoding domain may be one that encodes a deletion mutant product or a point mutation product.
• mutation of the PEST-like sequence encoding domain is one that disrupts a potential mitogen activated protein kinase (MAPK) phosphorylation site within the PEST-like sequence.
• MAPK mitogen activated protein kinase
• the mutation is one that encodes a point mutant at a residue from position 30 and 60.
• the residue that is substituted in the encoded mutant product is residue 44.
• the S at position 44 is substituted with a non-S residue, where the substituting residue may be A, G, I, F, C, L, M, V, etc, but is A in certain embodiments.
• the mutation is one that provides for more of the protein being produced. As such, in these embodiments there may not be a codon mutation that results in an altered residue, such as S44A, but instead results in a codon selection that provides for more RNA as compared to wildtype, and therefore ultimately more protein.
• the mutation of the PEST-like sequence-encoding domain is one that provides for a deletion of at least a portion of, if not all of, the residues that make up the PEST-like sequence.
• the mutation may be a deletion of one or more residues, including all of the residues, from about 30 to about 60, e.g., a deletion of residues 34 to 59.
• the attenuated bacteria of interest are not bacteria in which the entire PEST-like sequence has been deleted from the encoded LLO product, such as the bacteria reported in Decatur et al., Science (2000) 290:992-995.
• the subject attenuated bacteria have only a single type of hly mutation, as described above. In yet other embodiments, the bacteria have two or more of the specific hly mutations, as described above.
• the subject attenuated bacteria also find use as vaccines.
• the vaccines of the present invention are administered to a vertebrate by contacting the vertebrate with a sublethal dose of the attenuated Listeria vaccine, where contact typically includes administering the vaccine to the host.
• the attenuated bacteria are provided in a pharmaceutically acceptable formulation. Administration can be oral, parenteral, intranasal, intramuscular, intradermal, intraperitoneal, intravascular, subcutaneous, direct vaccination of lymph nodes, administration by catheter or any one or more of a variety of well- known administration routes. In farm animals, for example, the vaccine may be administered orally by incorporation of the vaccine in feed or liquid (such as water).
• E. coli expression strains were grown overnight in LB containing 30 ⁇ g/ml kanamycin (LB-KAN). 400 ⁇ l of the overnight culture was added to 10 ml LB-KAN and grown for 1.5 h, and then 1 mM IPTG was added. This culture was incubated at 30°C, shaking for 3 h. Cultures were pelleted and then resuspended in 1 ml storage buffer (140 mM sodium chloride, 4 mM potassium chloride, 10 mM sodium phosphate, 0.5 mM DTT, pH 6.0) with 1 mM PMSF. The samples were sonicated on ice and cleared by centrifugation.
• 1 ml storage buffer 140 mM sodium chloride, 4 mM potassium chloride, 10 mM sodium phosphate, 0.5 mM DTT, pH 6.0
• the lysate was centrifuged for 20 min at 17,000 g. The supernatant was collected and mixed into 5 ml Ni-NTA resin (QIAGEN) equilibrated in lysis buffer. The slurry was stirred at 4°C for 60 min to bind his-tagged protein to the resin. To remove unbound protein, the resin was packed into a column and washed with lysis buffer by dropwise gravity flow until UV absorbance of the eluate reached baseline, and then it was washed with wash buffer (lysis buffer, pH 6.0, 10% glycerol, 0.1% Tween 20).
• wash buffer lysis buffer, pH 6.0, 10% glycerol, 0.1% Tween 20.
• Washed resin was removed from the column, resuspended in elution buffer (lysis buffer, pH 6.0, and 800 mM imidazole), and incubated 10 min on ice, after which the supernatant was collected. This procedure was performed twice, yielding 6 ml eluate. Eluate was dialyzed in cassettes (Pierce Chemical Co.) within autoclaved storage buffer (lysis buffer, pH 6.0, with 1 mM EDTA). Both the Bradford method and UV280 absorbance determined protein concentrations. The procedure yielded ⁇ 25 mg protein per liter starting culture. Aliquots not used immediately were stored in storage buffer with 50% glycerol at -80°C.
• DNA fragment was produced with the method of splicing by overlap extension PCR, using the primers, templates, and restriction enzymes in Table 1 , and then ligated into the temperature-sensitive plasmid vector pKSV7. Allelic exchange was performed as described previously (Camilli, A., L.G. Tilney, and D.A. Portnoy. 1993. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8:143-157.). Strains were verified initially by detecting the loss of an .e1 site in a chromosomal PCR product containing the mutation.
• LD 50 by intravenous infection was established as previously described using BALB/c mice (Portnoy, D.A., P.S. Jacks, and D.J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167:1459-1471.).
• the percentage of bacteria that had escaped from the phagosome was determined by evaluating the presence of F-actin-coated bacteria within the macrophage, similar to an experiment previously described (Jones, S., and D.A. Portnoy. 1994a. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect. Immun. 62:5608-5613). C57/BL6 BM ⁇ s in DME, 10% FBS, with or without 0.5 ⁇ M bafilomycin A1 (Calbiochem), on a coverslip were infected for 15 min, resulting in a bacterium within 10% of the cultured macrophages.
• Macrophages were washed with Ringer's buffer (5 mM NaCI, 5 mM KCI, 2 mM CaCI 2 , 1 mM MgCI 2 , 2 mM NaH 2 P0 4 , 10 mM Hepes, 10 mM glucose, pH 7.2) and 25 ⁇ g/ml gentamicin was added.
• Ringer's buffer 5 mM NaCI, 5 mM KCI, 2 mM CaCI 2 , 1 mM MgCI 2 , 2 mM NaH 2 P0 4 , 10 mM Hepes, 10 mM glucose, pH 7.2
• the macrophages were fixed for 15 min with cytoskeletal fixative (40 mM Hepes, 10 mM EGTA, 0.5 mM EDTA, 5 mM MgS0 4 , 33 mM potassium acetate, 0.02% sodium azide, 5% polyethylene glycol 400, 4% paraformaldehyde), washed, permeabilized with PBS, containing 2% goat serum and 0.3% Triton X-100, and stained with Texas red-phalloidin (Molecular Probes) and DAPI (Molecular Probes). A total of 50 macrophages harboring bacteria were examined for each bacterial strain in each of four experiments.
• cytoskeletal fixative 40 mM Hepes, 10 mM EGTA, 0.5 mM EDTA, 5 mM MgS0 4 , 33 mM potassium acetate, 0.02% sodium azide, 5% polyethylene glycol 400, 4% paraformaldehyde
• phagosomal pH was performed essentially as previously described (Beauregard, K.E., K.D. Lee, R.J. Collier, and J.A. Swanson. 1997. pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J. Exp. Med. 186:1159-1163.) with the following modifications.
• fluid-phase fluorescein dextran molecular weight 10,000 (Molecular Probes) was added to the bacteria-containing media used to infect macrophages.
• Phagosomes containing both 10-kD fluorescein dextran and bacteria were photographed every 30 s with a Quantix cooled charge-couple device camera (Photometries) through fluorescent microscopy using a Nikon TE300 inverted microscope (Nikon), with phase-contrast and excitation wavelengths 485 and 440 nm and emission measurement at 520 nm. Images and the 485:440 ratio were collected until perforation was indicated by loss of dye from the vacuole. The 485:440 ratio measured just before perforation was compared with a standard curve to establish pH, as described in the published methods.
• BM0 were chosen for this assay because infected J774 are difficult to remove from tissue culture dishes without causing plasma membrane damage, whereas BM0 lift from the dish when incubated at 4°C.
• LLO Intracellular levels of LLO were studied with previously established methods (Moors, M.A., B. Levitt, P. Youngman, and D.A. Portnoy. 1999. Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect. Immun. 67:131-139.) and the following modifications.
• J774 cells were infected with L. monocytogenes strains for 30 min and then washed, and 50 ⁇ g/ml gentamicin was added at 60 min. 4 h after infection, methionine-starved cells were pulsed with [ 35 S]methionine (NEN Life Science Products) for 1 h.
• Plaquing assays within L2 cell monolayers were performed as described previously (Sun, A., A. Camilli, and D.A. Portnoy. 1990. Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell- to-cell spread. Infect. Immun. 58:3770-3778.), with modifications to the methods of measurement (Skoble, J., D.A. Portnoy, and M.D. Welch. 2000. Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J. Cell Biol. 150:527-538.).
• L2 cells were grown to confluency in six-well tissue culture dishes and then infected with bacteria for 1 h. Subsequently, DME-agar containing gentamicin was added and plaques were grown for 3 d. Living cells were visualized by adding on day 3 an additional DME- agar overlay containing neutral red (GIBCO BRL) and incubating overnight.
• DME-agar containing gentamicin was added and plaques were grown for 3 d. Living cells were visualized by adding on day 3 an additional DME- agar overlay containing neutral red (GIBCO BRL) and incubating overnight.
• the capacity of the LLO L461T mutant to grow in animals was evaluated by the lethal dose-50 (LD 5 o) in the mouse listeriosis model.
• the LD 5 o of the LLO L461T mutant was >3 x 10 6 as compared with an LD 50 of 1-3 x 10 4 for wild-type bacteria.
• the LLO L461T mutation does not affect the efficiency or pH of phagosomal escape Based on the observation that LLO has an acidic pH optimum and the bacteria escape from phagosomes at an acidic pH (Beauregard et al., Exp. Med. (1997) 186:1159-1163.), we hypothesized that a mutant LLO with greater activity at a neutral pH may act prematurely and not mediate escape efficiently.
• We used a fluorescence-based assay to monitor escape from the phagosome based on the observation that bacteria within the cytosol nucleate host actin filaments on their surface, whereas bacteria in vacuoles do not.
• LLO L461T mutation had no effect on phagosomal acidification or escape, and that phagosomal acidification was necessary for the escape of the LLO L461T mutant as well as for wild type. Therefore, it is unlikely that the LLO L461T mutant's virulence defect reflects a reduced ability to escape from phagosomes or an effect on phagosome maturation. The defect is likely due to the alteration of a different part of the pathogenic life cycle.
• the LLO L461T mutant had no defect in phagosomal escape, we next examined the capacity of the bacteria to grow in host cells using a quantitative tissue culture assay (Portnoy et al., 1988, supra). In this assay, adding the antibiotic gentamicin to the culture medium kills extracellular bacteria but has no measurable effect on the growth of intracellular wild-type bacteria. Between 2 and 5 h after infection, the LLO L461T mutant grewwell within J774 macrophages with an average apparent doubling time of 58 ⁇ 8 min, slightly longer than the wild-type doubling time of 42 ⁇ 4 min.
• the LLO L461T mutant grew with a nearly twofold longer average apparent doubling time (159 ⁇ 30 min compared with the wild-type doubling time of 83 ⁇ 8 min). Additionally, the LLO L461T mutant did not grow to as high a maximum number of bacteria.
• LLO L461T mutant has longer apparent doubling times and lower maximum bacterial numbers could reflect either a decrease in the overall growth rate or, more likely, an increase in the death of a subpopulation of intracellular bacteria.
• the analysis is complicated by the fact that after 5 h, L. monocytogenes spread from cell to cell. To eliminate cell-to-cell spread from the analysis, an in-frame deletion was introduced within the actA gene. The resulting strain was fully capable of vacuolar escape and intracellular growth within the original host cell, but was unable to nucleate actin filaments and thus unable to enter the secondary cell's double-membraned vesicle or spread from cell to cell.
• LDH lactate dehydrogenase
• permeabilization of the cell allowed the influx of gentamicin, which then killed the intracellular bacteria and prevented further permeabilization and LDH release.
• gentamicin was removed after 2 h, only the J774 cells infected with the LLO L461T ⁇ ActA mutant released high quantities of LDH.
• a monoclonal antibody that neutralizes LLO activity was added extracellularly to the J774 cells, there was no effect on LDH release, indicating that toxicity is mediated by intracellular LLO (unpublished data).
• a more sensitive method to test the integrity of the plasma membrane uses the membrane-impermeant dye propidium iodide.
• the dye enters the cell and increases its fluorescence upon binding cellular DNA. Staining can be measured by flow cytometry. After infection with the wild-type bacteria, most macrophage host cells still excluded the dye. In contrast, infection with the LLO L461T mutant led to permeabilization of about half of the macrophages, and infection with the LLO L461T ⁇ ActA mutant permeabilized most of the macrophages.
• the DP-L4017 strain expresses an LLO mutant which is 10-fold more hemolytic at neutral pH, relative to wild type LLO, which results in quicker damage to the host cell.
• This strain was also found to be 100-fold less virulent, by LD50 in BALB/c mice, and by 48 hours was 74 and 21 -fold less abundant in the spleen and liver, respectively, than wild type bacteria, in a competitive index assay. As such, the strain exhibits increased cytotoxity and decreased virulence as compared to wild type.
• the strain establishes an active infection in the mouse model that is limited by its cytotoxicity and cleared efficiently from the host system.
• the DP-L4384 strain contains both of the above described mutations, i.e., mutation S44A and mutation L461T) in LLO and was constructed using protocols analogous to those described above.
• the strain incorporates all of the properties of the above two described strains.
• strain DP-L4384 is 4.6 ⁇ 10 5 and 1.7 ⁇ 10 5 -fold less abundant than wild type bacteria in the spleen and liver, respectively, in a competitive index assay (Auerbuch, V. et al, supra). As such, the strain exhibits increased cytotoxity and decreased virulence as compared to wild type.
• the strain establishes an active infection in the mouse model that is limited by its cytotoxicity and cleared efficiently from the host system.
• the DP-L4042 was constructed as described in Decatur & Portnoy, Science (November 3, 2000) 290: 992-995.
• This strain contains a deletion of residues 34 to 59 of LLO, and therefore deletes the entire PEST-like sequence found at the N-terminus of LLO.
• the strain is extremely cytotoxic, and therefore is essentially undetectable in the competitive index assay after 48 hours.
• the strain has an LD 50 of 2 ⁇ 10 8 , approximately 10,000 times higher than the wild-type bacteria. As such, the strain exhibits increased cytotoxity and decreased virulence as compared to wild type.
• the strain establishes an active infection in the mouse model that is limited by its cytotoxicity and cleared efficiently from the host system.
• L. monocytogenes strains with deletions of actA were constructed by allelic exchange as described previously (Camilli, A., L.G. Tilney, and D.A. Portnoy. 1993. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol 8:143-157; Skoble, J., D.A. Portnoy, and M.D. Welch. 2000. Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and
• Tissue culture cells were grown in DMEM (Gibco-BRL) 7.5% heat deactivated fetal bovine serum (FBS)(Hy-Clone, Logan, UT) 2 mM glutamine
• Strain LLO S44A (DP-L4057) was produced using splicing by overlap extension PCR (Horton, R.M., Z.L. Cai, S.N. Ho, and L.R. Pease. 1990. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.
• phage U153 isolated from DP-L3903 were added to the recipient strain while in mid-log growth, 10 mM CaCI 2 and 10 mM MgCI 2 were added, and bacteria were incubated at room temperature for 1 hour, with occasional mixing. After one hour, 0.1 ⁇ g/ml erythromycin was added for 30 minutes, and then the mixture was spread on 1 ⁇ g/ml erythromycin BHI-agar plates and incubated at 37° for 2 days.
• L2 cell monolayers Plaquing assays within L2 cell monolayers were performed as described previously (Jones, S., and D.A. Portnoy. 1994, supra), with modifications to the methods of measurement (Skobel et al., supra). Briefly, L2 cells were grown to confluency in 6-well tissue culture dishes, and then infected with bacteria for 1 hour. Subsequently, DMEM agar containing 5 ⁇ g/ml gentamicin was added and plaques were grown for 3 days. Living cells were visualized by adding on day 3 an additional DMEM-agar overlay containing neutral red (Gibco-BRL) and incubating overnight.
• DMEM agar containing 5 ⁇ g/ml gentamicin
• Lethal Dose 50 determination was performed by Cerus Pharmaceuticals (Concord, CA) by tail vein injection in C57BL/6 mice as previously described (Portnoy et al., 1988, supra).
• Competitive indexes of LLO mutants, marked with erythromycin resistance, versus wild-type bacteria or single strain infections were performed essentially as previously described (Auerbuch, et al., supra , with the following modifications.
• Bacterial strains intended for injection into the mouse were grown in BHI until they reached an OD600 of 0.5, then 1 ml samples were frozen at -80° until subsequent use. These frozen samples were thawed and used to inoculate 10 ml of BHI, and grown at 37° until an OD of 0.5.
• Wild type mice were infected by tail vein injection of 5 x 10 5 CFU.
• RB6-8C5 monoclonal antibody treated were infected with 5 x 10 3 CFU, since a dose of 5 x 10 5 CFU lead to death before the 48-hour time point.
• 1 x 107 CFU were injected in the ⁇ ActA competitive index assay.
• the mutant bacteria were differentiated from the wild-type bacteria in the competitive index by treating organ lysates with 0.1 ⁇ g/ml erythromycin for 30 minutes to induce the resistance gene, then plating the sample on LB-agar plates and 1 ⁇ g/ml erythromycin BHI-agar plates to establish a ratio of sensitive (wild type) to resistant (mutant) bacteria at each respective time point.
• mice that were treated with RB6-8C5 were injected with 100 ⁇ g monoclonal antibody via the tail vein 6 hours before bacterial infection.
• Gentamicin-treated mice were injected with 1 mg Garamycin® (gentamicin sulfate, Schering Corporation, Kenilworth, NJ) in PBS subcutaneously six hours prior to organ harvest. 12 hours after injection we found the concentration of gentamicin to be 5.6 ⁇ g/ml in the pooled serum of 3 mice (performed by Debra Randall, Stanford University Hospital Clinical Labs, Palo Alto, CA), which is sufficient to inhibit bacterial growth.
• mice Mouse blood was removed by cardiac puncture on mice anesthetized with isofluorane (Abbott Labs, IL), then allowed to clot overnight at 4 ° . The clot was removed and the samples were centrifuged to allow separation of serum from any remaining solids. 1 x 10 3 bacteria were added to each sample of 50% serum- PBS, and then time points were taken by plating dilutions on LB-agar plates. Incubating the serum at 65° for 30 minutes produced heat-deactivated serum. 8. Tables
• Figure 3 We conclude that the more cytotoxic the strain of L. monocytogenes, the less virulent the strain is in the mouse model of listeriosis.
• a competitive index assay was performed with each mutant strain to establish a more accurate measurement of the mutants' virulence defects relative to the wild-type bacteria (Auerbuch, V., L. Lenz, and D. Portnoy. 2001. Developmment of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice. Infection and Immunity).
• a one-to-one ratio of wild-type bacteria and erythromycin (erm)-resistant mutants were coinjected into mice, and the ratio of wild-type bacteria to erm-resistant (mutant) bacteria was established in the spleen and liver.
• We found the trend for the defect in virulence to be similar to the LD 50 ( Figure 4), where the greater the cytotoxicity of the strain the fewer bacteria were recovered, relative to the wild-type bacteria.
• Granulocytes are a Major Contributor to the Cytotoxic Mutants' Growth Defect in Mice
• Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody. J Exp Med 179:259-268). In neutropenic mice the relative virulence defect of the cytotoxic mutants was eliminated 99%in the spleen, allowing the cytotoxic mutants to grow much more similarly to the coinjected wild-type bacteria in the competitive index assay (Figure 4). Less of an effect was observed in the liver, relative to the spleen, yet by 48 hours the more cytotoxic mutants' (LLO S44A and LLO S44A L461T) relative virulence increased 10-fold with the elimination of neutrophils. These data suggest that the cytotoxic mutants are more susceptible to neutrophil killing in immunocompetent mice. 4. A Larger Percentage of Cytotoxic Bacteria is Extracellular
• neutrophils were vital to limiting bacterial growth. However, they were more effective at controlling the cytotoxic mutants than wild- type bacteria, since their elimination allowed the cytotoxic bacteria to grow at a rate more similar to wild-type bacteria. We found that the cytotoxic mutants were less sensitive to the function of neutrophils in the liver than in the spleen. Based on our model, where cytotoxic mutants are exposed to extracellular defenses, there are a number of explanations that may account for these differences observed in different tissues. One possibility is that hepatocytes are capable of coping with cytotoxic bacteria better than splenic cells.
• neutrophils selectively lyse cells infected with cytotoxic bacteria.
• Previous publications reported that neutrophils are capable of lysing L. monocytogenes- infected hepatocytes, though the mechanism, whether direct or indirect, has not been established.
• the lysis-targeting signal received by neutrophils from infected cells may be elicited by cell damage. Since the cytotoxic bacteria damage the host cell, the cells infected by cytotoxic bacteria would thus be targeted for lysis earlier than the wild-type bacteria.
• Permeabilization of the host cell's plasma membrane may allow the efflux of activated complement or bacterial components, such as formylated peptides, that are chemoattractants of neutrophils that have been shown to be important in the mouse's resistance to L monocytogenes.
• activated complement or bacterial components such as formylated peptides
• the cytotoxic bacteria would be targeted for phagocytosis and destruction earlier than the wild- type bacteria, since they would emit chemotactic signals from within damaged cells.
• Exposure to the lytic functions of complement is unlikely to directly affect L. monocytogenes since we have described, in this study, that the bacteria grow at similar rates in normal of heat-deactivated mouse serum.
• the opsonizing properties of complement may act to target the cytotoxic bacteria for more efficient phagocytosis.
• cytotoxic mutants are a more visible threat than the wild-type bacteria.
• the bacteria may cause the liberation of more inflammatory cytokines, and thereby recruit more inflammatory cells to the foci of infection, as well as activate the function of those infiltrates.
• a number of inflammatory cytokines including TNF ⁇ , IFN ⁇ , IL-1 / ⁇ and IL-6, are vital for resistance to L monocytogenes.
• the greater presence of inflammatory cells, such as neutrophils, that are activated to a greater degree would then foster the greater clearance of the bacteria from the foci of infection.
• the antibiotic gentamicin has been used in both tissue culture and in vivo as a means to eliminate extracellular bacteria.
• our two most cytotoxic mutants, secreting LLO S44A or LLO S44A L461T were particularly sensitive to gentamicin injected into infected mice.
• this effect was only detectable when neutrophils were first eliminated.
• This finding indicates that the same population of bacteria that are sensitive to gentamicin are also sensitive to the activity of neutrophils.
• gentamicin did not entirely restore neutropenic mice to the level of resistance observed in immunocompetent mice.
• This finding indicates, in agreement with the rest of our data, that the virulence defect observed for the cytotoxic mutants is multifactoral.
• the lack of gentamicin's ability to completely replace the activity of neutrophils may indicate that neutrophils are playing a broader role in bacterial clearance than simply phagocytosing and destroying extracellular bacteria.
• a third class of mutants was hemolytic and prototrophic, and therefore likely contained transposon insertions in genes important specifically for intracellular growth; one of these mutants, DP-L2214, was selected for further analysis.
• DP-L2214 exhibited normal growth in both rich and minimal bacteriological media (data not shown).
• replication of DP-L2214 in J774 macrophages aborted at approximately 5 h.p.i.
• DP-L2214 has a replication defect that is restricted to the intracellular environment.
• AlplAI was also compared to DP-L2214 in a L2 fibroblast plaquing assay that measures intracellular growth over a 3 day infection. Both AlplAI and DP-L2214 exhibited plaque size that was 56 % and 58% of the wildtype plaque size respectively. In addition, we observed a unique mixed plaquing phenotype associated with both AlplAI and DP-L2214; the standard deviation from the average plaque size was 3 times greater in the mutants than in the wildtype strain.
• E. coli LplA ligates free lipoic acid to the E2 subunit of pyruvate dehydrogenase (PDH) and other structurally related enzymes(D. E. Brookfield, J. Green, S. T. Ali, R. S. Machado, J. R. Guest, FEBS Lett 295, 13-6 (Dec 16, 1991).
• PDH pyruvate dehydrogenase
• other structurally related enzymes D. E. Brookfield, J. Green, S. T. Ali, R. S. Machado, J. R. Guest, FEBS Lett 295, 13-6 (Dec 16, 1991).
• PDH pyruvate dehydrogenase
• other structurally related enzymes D. E. Brookfield, J. Green, S. T. Ali, R. S. Machado, J. R. Guest, FEBS Lett 295, 13-6 (Dec 16, 1991).
• BHI brain-heart infusion
• E2 subunit of pyruvate dehydrogenase by mass spectroscopy. No difference in lipoylation of E2 PDH was observed between the wildtype and the ⁇ /p/A " strain. These data identify E2 PDH as a major target of lipoic acid modification in L. monocytogenes, consistent with reported observations in E. coli.
• total cell lysates were prepared from intracellular bacteria for SDS-PAGE and Western blot analysis. Equivalent loading of bacterial proteins was confirmed by Western Blot analysis of an unrelated protein, ActA. While lipoylated E2 PDH was observed in wildtype bacteria grown in macrophages, the modified form of E2 PDH was not present in AlplAI lysates.
• the pool of modified E2 PDH present in the bacterial innoculum after overnight culture in rich media may have allowed the AlplAI strain to undergo approximately 4 rounds of cell division in the host cell over 5 hrs before depleting functional E2 PDH. If lipoylated E2 was depleted after several rounds of cells division, AlplAI mutant bacteria isolated from host cells should not be able to establish a productive infection. We isolated wildtype and mutant bacteria from infected macrophages 4 h.p.i. and used these bacteria to infect a new monolayer of macrophages. During the subsequent infection the wildtype strain grew very aggressively while the AlplAI strain did not replicate at all.
• IplAi performs a critical and non-redundant function during intracellular growth that involves modification of E2 pyruvate dehydrogenase.
• Lipoic acid has been shown to have anti-oxidant properties in mammalian cells (L. Packer, Drug Metab Rev 30, 245-75 (May, 1998)). Therefore, we considered the possibility that lipoic acid in L. monocytogenes as part of the PDH complex might also act to protect the bacteria from oxidative stress in the host cell.
• Host cells may have several sources of oxidative stress. First, macrophages are able to produce reactive oxygen and nitrogen intermediates in response to phagocytosis. Secondly, all cells produce reactive oxygen species as a normal by-product of oxidative metabolism.
• the macrophages were subjected to TUNEL staining.
• TUNEL positive bacteria we were able to observe TUNEL positive bacteria in macrophages infected by the ⁇ /p/A mutant strain, but not macrophages infected by wildtype bacteria.
• TUNEL positive bacteria was rare, they occurred in clusters, such that the TUNEL positive bacteria in one cluster were contained within one host cell.
• the presence of DNA strand breaks in AlplA "/mutant bacteria supports the hypothesis that E2-lipoamide protects bacteria against oxidative stress.
• E. coli also has two lipoate protein ligases, LplA and LipB, that transfer lipoic acid from different sources to E2 PDH (T. W. Morris, K. E. Reed, J. E. Cronan, Jr., J Bacteriol 177, 1-10 (Jan, 1995); K. E. Reed, J. E.
• E. coli LipB utilizes de novo synthesized lipoic acid from octanoyl-acyl carrier protein.
• E. coli LplA ligates scavenged free lipoic acid to E2 PDH.
• L. monocytogenes is a lipoic acid auxotroph, and uses lipoic acid scavenged from its environment; maintenance of two lipoate protein ligase genes in the genome implies different external sources of lipoic acid.
• Studies of lipoic acid metabolism in mammalian cells suggest that very little free lipoic acid is present in the cytosol under normal physiological conditions. Thus, we hypothesize that in L.
• E2 PDH is the primary target of LplA1 in L. monocytogenes.
• Previous research has focused on the function of PDH in intermediary metabolism in converting pyruvate into acetyl CoA which represents the entry of carbon into the tricarboxylic acid cycle.
• the metabolic function of PDH which requires the E1 , E2 and E3 subunits, is important for aerobic growth, and lack of PDH enzymatic activity is likely responsible for the abortive growth phenotype we have observed in the AlplAI mutant strain.
• recent studies have revealed novel functions for the E2 subunit of PDH that appear independent of the PDH holoenzyme.
• E2 and E3 required for the redox capacity of lipoamide, contribute to the reducing capacity of a protein complex isolated from Mycobacterium tuberculosis extracts. This reducing activity may mediate M. tuberculosis resistance to oxidative stress in vivo.
• Our data showing that the AlplA "/strain is more susceptible to oxidative stress are consistent with this hypothesis.
• studies in Pseudomonas aeruginosa, B. subtilis, and ⁇ . thuringensis have revealed a role for E2 PDH in DNA binding and/or transcriptional regulation. The requirement for lipoamide modification in those processes is not known.
• mice are immunized with 0.1 LD 50 of LM-NP. Two weeks later, the mice are sacrificed and primary cultures are set up of spleen cells with either influenza infected (A/PR8/34) splenocytes or with a synthetic peptide 147-158 known to represent the immunodominant epitope of the NP protein. After four days in culture, the cytolytic activity of both populations is measured against CT26-NP, RENCA-NP and the parental cell lines CT26 and RENCA. A positive control is included (P815, a mastocytoma tumor cell line known to be efficiently lysed by H- 2 d restricted CTL in the presence of the peptide or when infected by A PR8/34).
• a positive control is included (P815, a mastocytoma tumor cell line known to be efficiently lysed by H- 2 d restricted CTL in the presence of the peptide or when infected by A PR8/34).

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